Project Summary/Abstract Objectives and Rationale: Our long-term goal is to engineer synthetic oscillators or switches that can test key design principles of endogenous biological pathways and correct the pathways when they are mis-regulated. Many mechanisms are involved in the mammalian circadian oscillator, but their cyclic nature and interconnections make it very difficult to test which mechanisms are truly essential. Building a synthetic circadian clock in mammalian cells using similar design principles as the endogenous ones would be the most direct and convincing way to test and identify key mechanisms. This innovative construct would also enable us to understand and develop new treatments for circadian sleep disorders and other types of neurological or physiological dysfunction caused by faulty clocks. Based on mathematical modeling and past experiments, the unique parameters of the rate-limiting clock component PER protein are absolutely required to build a synthetic circadian clock. The central hypothesis here is that many heterologous transcriptional feedback loops can produce autonomous circadian rhythms if the feedback inhibition is mediated by PER protein because PER can generate necessary circadian parameters such as time delay and nonlinearity. Aim 1. Generate a fully synthetic circadian clock in mammalian cells. A synthetic transcriptional feedback loop will be generated using the yeast-derived GAL4-UAS-GAL80 system: GAL4 will activate transcription of UAS-Gal80 but later will be inhibited by GAL80 to close the feedback loop. PER will be fused to GAL80 to ensure?if our hypothesis is correct?that this feedback inhibition is mediated in a circadian manner. It has been demonstrated in several cases that the circadian activities of PER are not affected by fusion with other proteins such as Luciferase and Venus. The functionality of the synthetic oscillator will be assessed by measuring rhythms from UAS-Luciferase or GFP in real time, as it has been done previously. According to mathematical predictions, the circuit will produce robust circadian rhythms once it is reset by temporarily inducing Per2-Gal80 expression from a second, drug-inducible promoter. Aim 2. Identify a novel motif from the PER2 protein critical for the 24-hour oscillations. Our preliminary data suggest that the circadian rhythmicity of PER2 is dependent upon a specific domain of PER2 that is subjected to unique posttranslational regulation. Degrons have been engineered into synthetic circuits to provide enhanced degradation without specific time kinetics. We believe that the ?24-hour domain? identified by this study could be engineered into other proteins and circuits to confer a circadian property to the circuits, without incorporating the full-length PER protein. Furthermore, this motif and the mechanisms that act upon it would be a prime target for therapeutics to modulate the circadian clock.